The effect of adenosine A(2A) receptor antagonists on hydroxyl radical, dopamine, and glutamate in the striatum of rats with altered function of VMAT2.

Abstract

It has been shown that a decreased vesicular monoamine transporter (VMAT2) function and the disruption of dopamine (DA) storage is an early contributor to oxidative damage of dopamine neurons in Parkinson's disease (PD). In our previous study, we demonstrated that adenosine A(2A) receptor antagonists suppressed oxidative stress in 6-hydroxydopamine-treated rats suggesting that this effect may account for neuroprotective properties of drugs. In the present study, rats were injected with reserpine (10 mg/kg sc) and 18 h later the effect of the adenosine A(2A) receptor antagonists 8-(3-chlorostyryl)caffeine (CSC) and 4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethyl)phenol (ZM 241385) on extracellular DA, glutamate and hydroxyl radical formation was studied in the rat striatum using in vivo microdialysis. By disrupting VMAT2 function, reserpine depleted DA stores, and increased glutamate and hydroxyl radical levels in the rat striatum. CSC (1 mg/kg) but not ZM 241385 (3 mg/kg) increased extracellular DA level and production of hydroxyl radical in reserpinised rats. Both antagonists decreased the reserpine-induced increase in extracellular glutamate. L-3,4-Dihydroxyphenylalanine (L-DOPA) (25 mg/kg) significantly enhanced extracellular DA, had no effect on reserpine-induced hydroxyl radical production and decreased extracellular glutamate concentration. CSC but not ZM 241385 given jointly with L-DOPA increased the effect of L-DOPA on extracellular DA and augmented the reserpine-induced hydroxyl radical production. CSC and ZM 241385 did not influence extracellular glutamate level, which was decreased by L-DOPA. It seems that by decreasing the MAO-dependent DA metabolism rate, CSC raised cytosolic DA and by DA autoxidation, it induced hydroxyl radical overproduction. Thus, the methylxanthine A(2A) receptor antagonists bearing properties of MAO-B inhibitor, like CSC, may cause a risk of oxidative stress resulting from dysfunctional DA storage mechanism in early PD.

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The Effect of Adenosine A2AReceptor Antagonists on Hydroxyl
Radical, Dopamine, and Glutamate in the Striatum of Rats
with Altered Function of VMAT2
Krystyna Gołembiowska•Anna Dziubina
Received: 30 December 2011/Revised: 20 February 2012/Accepted: 22 February 2012/Published online: 10 March 2012
? The Author(s) 2012. This article is published with open access at Springerlink.com
Abstract
monoamine transporter (VMAT2) function and the dis-
ruption of dopamine (DA) storage is an early contributor to
oxidative damage of dopamine neurons in Parkinson’s
disease (PD). In our previous study, we demonstrated that
adenosine A2Areceptor antagonists suppressed oxidative
stress in 6-hydroxydopamine-treated rats suggesting that
this effect may account for neuroprotective properties of
drugs. In the present study, rats were injected with reser-
pine (10 mg/kg sc) and 18 h later the effect of the adeno-
sine A2A receptor antagonists 8-(3-chlorostyryl)caffeine
(CSC) and 4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a]
[1,3,5]triazin-5-ylamino]ethyl)phenol (ZM 241385) on
extracellular DA, glutamate and hydroxyl radical formation
was studied in the rat striatum using in vivo microdialysis.
By disrupting VMAT2 function, reserpine depleted DA
stores, and increased glutamate and hydroxyl radical levels
in the rat striatum. CSC (1 mg/kg) but not ZM 241385
(3 mg/kg) increased extracellular DA level and production
of hydroxyl radical in reserpinised rats. Both antagonists
decreased the reserpine-induced increase in extracellu-
lar glutamate.
L-3,4-Dihydroxyphenylalanine (L-DOPA)
(25 mg/kg) significantly enhanced extracellular DA, had
no effect on reserpine-induced hydroxyl radical production
and decreased extracellular glutamate concentration. CSC
but not ZM 241385 given jointly with L-DOPA increased
the effect of L-DOPA on extracellular DA and augmented
the reserpine-induced hydroxyl radical production. CSC
and ZM 241385 did not influence extracellular glutamate
It has been shown that a decreased vesicular
level, which was decreased by L-DOPA. It seems that by
decreasing the MAO-dependent DA metabolism rate, CSC
raised cytosolic DA and by DA autoxidation, it induced
hydroxyl radical overproduction. Thus, the methylxanthine
A2A receptor antagonists bearing properties of MAO-B
inhibitor, like CSC, may cause a risk of oxidative stress
resulting from dysfunctional DA storage mechanism in
early PD.
Keywords
antagonists ? VMAT2 function ? Oxidative stress
Parkinson’s disease ? Adenosine A2Areceptor
Introduction
Progressive degeneration of the dopamine-containing
neurons in the substantia nigra pars compacta results in
deficiency of striatal dopamine (DA) and loss of neuro-
chemical transport systems, such as the dopamine trans-
porter (DAT) and the vesicular monoamine transporter
(VMAT2) (Miller et al. 1997, 1999). PET studies in a non-
human primate model of Parkinson’s disease (PD) showed
that a decreased VMAT2 function and the disruption of DA
sequestration was an early and potent contributor to oxi-
dative damage of dopamine neurons in PD pathogenesis
(Chen et al. 2008).
DA is a highly reactive molecule that is capable of
autoxidation to a quinone in the basic pH of the cytosol.
Furthermore, cytosolic MAO-dependent DA metabolism
leads tothe formationofaldehydes and peroxides(Halliwell
2006). Regarding the role of oxidative stress in the patho-
genesis of PD, packing of cytosolic DA into synaptic vesi-
cles by VMAT2 prevents its autoxidation and subsequent
degeneration of dopamine neurons. An animal model of PD
mimicking the altered DA homeostasis by impaired DA
K. Gołembiowska (&) ? A. Dziubina
Institute of Pharmacology, Polish Academy of Sciences,
31-343 Krako ´w, 12 Sme ˛tna Street, Krako ´w, Poland
e-mail: nfgolemb@cyf-kr.edu.pl
URL: WWW.if.pan.krakow.pl
123
Neurotox Res (2012) 22:150–157
DOI 10.1007/s12640-012-9316-9

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storage mechanisms is based on the administration of the
irreversible VMAT2 inhibitor reserpine to rats (Carlsson
et al. 1957). Reserpine reduces vesicular storage and release
of brain monoamines and leads to the accumulation of oxi-
dative products of neurotransmitters (Caudle et al. 2007).
Recently, antagonists of adenosine A2A receptors
appeared as a new promising non-dopaminergic therapy of
PD. Striatopallidal neurons are highly enriched in adeno-
sine A2A receptors which occur there as heteromeric
complexes with dopamine D2receptors (Ferre ´ et al. 1993).
Behavioral studies in rodents and in non-human primates
showed that A2A receptor antagonists reversed motor
impairment induced by 6-hydroxydopamine (6-OHDA) or
MPTP (Schwarzschild et al. 2006; Morelli et al. 2007). The
mechanism of antiparkinsonian effects of A2A receptor
antagonists is based on their ability to modulate GABA
release and DA-dependent c-fos activation in the indirect
striatopallidal pathway (Pollack and Fink 1995; Ochi et al.
2000). In addition, presynaptic A2Areceptors are able to
control corticostriatalglutamatergic
counteracting D2 receptor function (Tozzi et al. 2007).
Several epidemiological and animal studies have suggested
neuroprotective effects of caffeine and selective A2A
adenosine receptor antagonists (Ross et al. 2000; Ascherio
et al. 2001; Xu et al. 2005; Chen et al. 2007). A protective
effect of caffeine and more selective antagonists of A2A
receptors, similar to genetic inactivation of A2Areceptors,
was observed in an animal MPTP neurotoxicity model (Xu
et al. 2005; Chen et al. 2007) or in ischemia and excitotoxic
brain injury models (Popoli et al. 2004; Chen et al. 2007).
The mechanism allowing A2A receptor antagonists to
protect dopaminergic neurons has not been fully explained
yet, but a variety of their effects on various types of neu-
rons, e.g., glutamatergic nerve terminals and glial or
immune cells, suggest its complex nature (Chen et al.
2007). In our earlier study, we have shown that A2A
receptor antagonists decreased the production of free rad-
ical and lowered extracellular glutamate level in 6-OHDA-
treated rats (Gołembiowska et al. 2009; Gołembiowska and
Dziubina 2012). Moreover, A2A receptor antagonists
administered in combination with L-3,4-Dihydroxyphenyl-
alanine (L-DOPA) did not change inhibitory effect of
L-DOPA on free radical generation and glutamate
enhancement in the striatum of 6-OHDA-treated rats
(Gołembiowska and Dziubina 2012).
A class of A2Aantagonists belonging to methylxanthine
derivatives offers a neuroprotective benefit as MAO-B
inhibitors (Castagnoli et al. 2003). Inhibition of DA deg-
radation by MAO-B attenuates hydrogen peroxide forma-
tion, but at the same time it increases the risk of DA
autoxidation resulting from augmentation of the cytosolic
DA pool. Therefore, in our present study, we aimed to
investigate whether two A2A receptor antagonists 8-(3-
transmissionby
chlorostyryl)caffeine
furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethyl)
phenol (ZM 241385) belonging to different chemical
classes (methylxanthine and non-xanthine derivatives,
respectively) can modulate extracellular level of DA and
glutamate as cellular sources of hydroxyl radical in animals
with reduced VMAT2 function after reserpine administra-
tion. Since L-DOPA may be toxic in the brain by pro-
moting the formation of reactive species and neurotoxic
quinones when cytosolic level of DA increases after dis-
ruption of DA storage mechanisms (Halliwell 2006), we
also studied the effect of A2Areceptor antagonists given in
combination with L-DOPA in rats treated with reserpine.
(CSC)and 4-(2-[7-amino-2-(2-
Materials and Methods
Animals
Microdialysis studies were conducted in male Wistar rats
(250–300 g), bred at the Institute of Pharmacology, Polish
Academy of Sciences, Krakow, Poland. The rats were
housed in temperature- and humidity-controlled rooms on a
12-h light/dark cycle, with free access to filtered tap water
and standard pelleted laboratory chow throughout the
study. The experimental procedures and housing conditions
used were in strict accordance with the Polish legal regu-
lations concerning experiments on animals (Dz. U.
05.33.289). All the experimental protocols were approved
bythe Local Bioethics
Experiments.
Commission forAnimal
Drugs
L-DOPA, CSC, benserazide, and p-hydroxybenzoic acid
(PBA) were obtained from Sigma-Aldrich (Poznan ´, Poland),
reserpine was obtained from Fluka-Analytical, Poland),
whereas4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]
triazin-5-ylamino]ethyl)phenol (ZM 241385) came from
TOCRIS (Warsaw, Poland). All the chemicals used for
HPLC were purchased from Merck (Warsaw, Poland).
L-DOPA and benserazide were dissolved in saline. A solu-
tion of PBA was prepared in an artificial cerebrospinal fluid
(aCSF) and was then adjusted to pH 7.4 with 0.1 M NaOH.
CSC was initially dissolved in dimethyl sulfoxide (DMSO;
Sigma-Aldrich, Poznan ´, Poland) and was then diluted in at
least 20 vols. of the vehicle consisting of a 20:80 (v/v)
mixture of Alkamulus EL-620 (Rhone-Poulenc, Cranbury,
NJ) and a phosphate-buffered saline. ZM 241385 was dis-
solved in a small amount of DMSO and then was diluted in
Cremophor EL (Sigma-Aldrich, Poznan ´, Poland) and 0.9%
NaCl (final concentration: a 15% DMSO and a 15% Crem-
ophor EL). All injections were made by an intraperitoneal
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route (i.p.). Reserpine dissolved in a mixture of benzyl
alcohol (2 ml), citric acid (250 mg), and Tween-80 in H2O
was given in a dose of 10 mg/kg 18 h before microdialysis
experiment.CSC(1 mg/kg)andZM241385(3 mg/kg)were
giveninsingleinjectionsasindicatedinthefigures.L-DOPA
(25 mg/kg) was injected 20 min after the administration
of A2A receptor antagonists together with benserazide
(12.5 mg/kg). Control animals received respective vehicles.
Determination of Monoamine Levels
For the measurement of dopamine (DA), 3,4-dihydroxy-
phenylacetic acid (DOPAC) and homovanilic acid (HVA),
the brain tissue was homogenized in 0.1 M HClO4, cen-
trifuged at 4?C for 5 min at 10,0009g, and the supernatant
was filtered through 0.1 lm (Millipore) membranes. An
aliquot of 2–5 ll of each sample was then injected into a
high performance liquid chromatograph (HPLC) with
electrochemical detection.
In Vivo Microdialysis
The rats were anaesthetized with ketamine (75 mg/kg i.m.)
and xylazine (10 mg/kg i.m.) and placed in a stereotaxic
apparatus (David Kopf Instruments, Tujunga, CA, USA).
Their skulls were exposed and small holes were drilled for
the insertion of microdialysis probes into the striatum using
the following coordinates: 1.8 mm anterior from the
bregma; 2.8 mm lateral from the sagittal suture; -7.0 mm
ventral from the dura (Paxinos and Watson 1998). Vertical
microdialysis probes were constructed as described in
detail elsewhere (Gołembiowska et al. 2009). Probe inlets
were connected to a syringe pump (BAS, IN, USA) which
delivered an aCSF composed of [mM]: NaCl 147, KCl 4.0,
MgCl21.0, CaCl22.2; pH 7.4 at a flow rate of 2 ll/min. All
metal parts of the aCSF delivery system were replaced with
PEEK components or were passivated with 6 M HNO3.
Baseline samples were collected every 20 min after the
washout period to obtain a stable extracellular neuro-
transmitter level. Appropriate drugs were then adminis-
tered 20 min before L-DOPA injection given at time 0, as
shown in figures, and dialysate fractions were collected for
240 min. At the end of the experiment, the rats were killed
and their brains were histologically examined to validate
probe placement.
Analytical Procedure
DA, DOPAC, and HVA were analyzed by HPLC with an
electrochemical detection. The level of hydroxyl radicals
was estimated as 3,4-dihydroxybenzoic acid (3,4-DHBA),
a product of the spin trap reagent PBA (1 mM) applied via
the microdialysis probe. DA and its metabolites were
simultaneously determined in the same fractions of striatal
dialysates. Chromatography was performed using an LC-
10 AD pump (Shimadzu Europa GmbH, Warsaw, Poland),
an LC-4B amperometric detector with a cross-flow detector
cell (BAS, IN, USA) and a BDS-Hypersil C18 analytical
column (3 9 100 mm, 3 lm; Thermo Electron Corp.,
UK). The mobile phase consisted of 0.1 M monochloro-
acetic acid adjusted to pH 3.7 with 3 M sodium hydroxide,
0.5 mM EDTA, 13 mg/l 1-octanesulfonic acid sodium salt,
a 5.7% methanol, and a 0.8% acetonitrile. The flow rate
was 0.5 ml/min, and the applied potential of a 3-mm glassy
carbon electrode was ?600 mV at a sensitivity of 2 nA/V.
Concentrations of all compounds were calculated by
comparing their peak areas with respective standards and
were processed by Chromax 2001 (Pol-Lab, Warsaw,
Poland) software run on a personal computer. The obtained
values were not corrected for in vitro probe recovery,
which was approximately 10–15%.
Glutamate was measured in dialysates (20 ll) after
derivatization with 4-dimethylaminoazobenzene-40-sulfo-
nylchloride (DABS-Cl) at 70?C for 12 min, according to
Knecht and Chang (1986). Dabsylated amino acids were
separated on an Ultrasphere ODS (4.6 9 150 mm, 3 lm)
column (Supelco, Poznan ´, Poland) by gradient elution,
with solvent A (10 mM citric acid, 4% dimethylformam-
ide) and solvent B (acetonitrile). Dabsylated compounds
were detected by measuring an absorbance at 436 nm using
Beckman Amino Acid System Gold with VIS detection.
Data Analysis
All obtained data are given in absolute numbers. The
statistical significance of differences between experimen-
tal groups was calculated using a one-way ANOVA for
repeated-measures, followed by Tukey’s post hoc test.
The results were considered statistically significant at
P\0.05.
Results
The Effects of Reserpine on DA, DOPAC, HVA
in the Rat Striatum
Reserpine produced a substantial depletion of DA and
changed the level of its metabolites DOPAC and HVA 24 h
after the injection (Table 1). Dialysate level of DA was
decreased by ca. 96%, while DOPAC and HVA extracel-
lular concentrations were increased by 44 and 11% of
control, respectively. The content of striatal DA was
attenuated by ca. 95%, while DOPAC and HVA contents
152 Neurotox Res (2012) 22:150–157
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were increased by 33 and 58%, respectively. These results
indicate reserpine-induced damage of intracellular DA
stores and increase in DA turnover.
The Effects of CSC and ZM 241385 on Extracellular
Level of DA, Glutamate and Production of Hydroxyl
Radical in Reserpine-Treated Rats
CSC (1 mg/kg) increased, while ZM 241385 (3 mg/kg) did
not influence extracellular level of DA in the rat striatum
attenuated by reserpine (Fig. 1a). Repeated- measures
ANOVA showeda significant
(F3,15= 37.63, P = 0), but a non-significant effect of time
(F14,210= 1.53, P = 0.103), and no interaction between
both factors (F42,210= 1.099, P = 0.33). Post hoc analysis
with Tukey’s test showed that CSC significantly increased
extracellular DA level from 20 to 200 min (P\0.05–0.01
in comparison with reserpine).
Hydroxyl radical production was increased in the stria-
tum of reserpinized rats (Fig. 1b). CSC (1 mg/kg), but not
ZM 241385 (3 mg/kg), further increased its production
(Fig. 1b). Repeated-measures ANOVA showed a signifi-
cant effect of treatment (F3,18= 24.21, P = 0.00001),
time (F11,198= 22.56, P = 0), and an interaction between
both factors (F33,198= 5.27, P = 0). Post hoc analysis
with Tukey’s test showed that CSC significantly increased
production of hydroxyl radical from 20 to 240 min after
the administration (P\0.05–0.01 in comparison with
reserpine).
Extracellular glutamate level was increased by reserpine
in comparison with control group (Fig. 1c). CSC (1 mg/kg)
and ZM 241385 (3 mg/kg) decreased the reserpine-
enhanced extracellular glutamate level, to control values
(Fig. 1c). Repeated-measures ANOVA showed a signifi-
cant effect of treatment (F3,12= 6.79, P = 0.006), time
(F14,168= 4.46, P = 0.00001) and an interaction between
both factors (F42,168= 1.97, P = 0.001). Post hoc analysis
with Tukey’s test showed that CSC and ZM 241385 sig-
nificantly decreased extracellular glutamate level from 40
effectof treatment
Table 1 Tissue content and
dialysate level of DA, DOPAC,
and HVA in the striatum of
control and reserpine
(10 mg/kg)-treated rats
* P\0.01 versus control
Mean ± SEM (n)
DA (pg/10 ll) DOPAC (ng/10 ll) HVA (ng/10 ll)
Dialysate
Control10.5 ± 0.53 (43)2.8 ± 0.11 (39) 1.9 ± 0.05 (39)
Reserpine0.4 ± 0.06 (44)*4.0 ± 0.07 (36)*2.11 ± 0.06 (39)*
% of control3.9 144111
DA (ng/mg wt) DOPAC (ng/mg wt) HVA (ng/mg wt)
Tissue
Control 12,442 ± 956 (12)1,331 ± 76 (12) 870 ± 118 (12)
Reserpine568 ± 99 (12)* 1,773 ± 327 (12)* 1,378 ± 281 (12)*
% of control5 133158
^
^
^
^
^
^^
^
^
^
^
0,1
0,2
0,3
0,4
0,5
0,6
0,7
-40 -200 20 406080 100 120 140 160 180 200 220 240
min
DA (pg/10 ul)
RES
RES + CSC 1 mg/kg
RES + ZM 3 mg/kg
0
5
10
15
-40 -200 20 406080 100 120 140 160 180 200 220 240
min
DA (pg/10 ul)
control
**
^^
^^
^^
^^
^^
^^
^^
^^
^^
^^
^
^^
0
500
1000
1500
2000
2500
-40 -200 20 40 6080 100 120 140 160 180 200 220 240
min
3,4-DHBA (pg/10 ul)
control
RES
RES + CSC 1 mg/kg
RES + ZM 3 mg/kg
0
1
2
3
4
5
6
-40 -20020 40 6080 100 120 140 160 180 200 220 240
min
GLU (pmoles/10 ul)
control
RES
RES + CSC 1 mg/kg
RES + ZM 3 mg/kg
^^
b
c
*
*
a
Fig. 1 The effects of CSC (1 mg/kg) and ZM 241385 (3 mg/kg) on
extracellular concentrations of DA (a), hydroxyl radical (3,4-DHBA,
b) and glutamate (GLU, c) in the striatum of rats treated with
reserpine (10 mg/kg). The injection of drugs is indicated by an arrow.
The data are the mean ± SEM (n = 5–7). *P\0.05; **P\0.01
versus control;^P\0.05;^^P\0.01 versus reserpine
Neurotox Res (2012) 22:150–157153
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to 240 min after administration (P\0.01 in comparison
with reserpine).
The Effect of L-DOPA on Extracellular Level of DA,
DOPAC, HVA, Glutamate and Production of Hydroxyl
Radical in Reserpine-Treated Rats
L-DOPA (25 mg/kg) increased the level of DA, DOPAC,
and HVA in the striatum of reserpinized rats (Figs. 2a, 3a,
b). Repeated-measures ANOVA showed a significant effect
of treatment on DA (F2,11= 30.55, P = 0.00003), time
(F14,154= 2.34, P = 0.006), but no interaction for both
factors (F28,154= 1.49, P = 0.07). Post hoc analysis with
Tukey’s test showed that L-DOPA significantly increased
extracellular DA concentration (P\0.05–0.01) in com-
parison with reserpine. Repeated- measures ANOVA
showed a significant effect of treatment on DOPAC
(F2,14= 20.66,
P = 0.0001), time(F14,196= 19.75,
P = 0) and interaction for both factors (F28,196= 18.85,
P = 0). Post hoc analysis with Tukey’s test showed that
L-DOPA significantly increased extracellular DOPAC
(P\0.01) in comparison with reserpine. Repeated-mea-
sures ANOVA showed a significant effect of treatment on
HVA (F2,14= 18.71, P = 0.0001), time (F14,196= 12.9,
P = 0), and interaction for both factors (F28,196= 18.43,
P = 0). Post hoc analysis with Tukey’s test showed that
L-DOPAsignificantlyincreased
(P\0.01) in comparison with reserpine.
Hydroxyl radical production, increased by reserpine,
was not changed in rats treated with L-DOPA (Fig. 2b).
Repeated-measures ANOVA showed a significant effect of
treatment (F2,13= 4.47, P = 0.03), time (F11,143= 19.55,
P = 0) andan interaction
(F22,143= 1.71, P = 0.03). Post hoc analysis with Tukey’s
test showed a significant difference (P\0.01) between
control and reserpine or control and a combination of
reserpine and L-DOPA (Fig. 2b).
Extracellular glutamate concentration, increased by
reserpine, was decreased by L-DOPA to the control level
(Fig. 2c). Repeated-measures ANOVA showed a signifi-
cant effect of treatment (F2,10= 7.52, P = 0.01), there
wasno significanteffect
P = 0.23) and there was an interaction between both fac-
tors (F28,140= 1.73, P = 0.02). Post hoc analysis with
Tukey’s test showed a significant effect of L-DOPA on
extracellular glutamate level (P\0.01).
extracellularHVA
betweenbothfactors
of time(F14,140= 1.27,
0
200
400
600
800
1000
-40 -200 204060 80 100 120 140 160 180 200 220 240
min
3,4-DHBA (pg/10 ul)
control
RES
RES+L-DOPA 25 mg/kg
**
b
0
1
2
3
4
5
6
-40 -200 20 40 60 80 100 120 140 160 180 200 220 240
min
GLU (pmoles/10 ul)
control
RES
RES+L-DOPA 25 mg/kg
^^
c
**
^
^
^^
^^
^^^^
^
^^
^^
^^
0
2
4
6
8
10
12
14
-40 -200 2040 6080 100 120 140 160 180 200 220 240
min
DA (pg/10 ul)
control
RES
RES+L-DOPA 25 mg/kg
**
a
Fig. 2 The effect of L-DOPA (25 mg/kg) on extracellular concen-
trations of DA (a), hydroxyl radical (3,4-DHBA, b) and glutamate
(GLU, c) in the striatum of rats treated with reserpine (10 mg/kg).
The injection of L-DOPA is indicated by an arrow. The data are the
mean ± SEM (n = 5–7). **\0.0P1 versus control;
^^P\0.01 versus reserpine
^P\0.05;
0
2
4
6
8
10
-40 -200 20 406080 100 120 140 160 180 200 220 240
min
HVA (ng/10 ul)
control
RES
RES+L-DOPA 25 mg/kg
b
**
^^
0
2
4
6
8
10
12
-40 -200 2040 60 80 100 120 140 160 180 200 220 240
min
DOPAC (ng/10 ul)
control
RES
RES+L-DOPA 25 mg/kg
**^^
a
*
Fig. 3 The effect of L-DOPA (25 mg/kg) on extracellular concen-
trations of DOPAC (a), and HVA (b) in the striatum of rats treated
with reserpine (10 mg/kg). The injection of L-DOPA is indicated by
an arrow. The data are the mean ± SEM (n = 5–7). *P\0.05;
**P\0.01 versus control;^^P\0.01 versus reserpine
154Neurotox Res (2012) 22:150–157
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